Oxidative stress plays an important role in the pathophysiology of a variety of cardiovascular diseases including ischemia-reperfusion injury following a myocardial infarction, heart failure, diabetic cardiomyopathy, and doxorubicin-induced cardiotoxicity. Small molecule and protein thiols as well as enzymes related to thiol metabolism have been shown to have an important role in the protection of the heart against oxidative stress processes. Because of the important role of thiols in the development of cardiovascular diseases, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as the role of medications in disease treatment. In this project, a novel glutathione reductase inhibitor, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio- carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), will be evaluated as a research tool to induce thiol oxidative stress in H9c2 rat cardiomyocytes and a mouse model. The specific aims of the proposed project are to evaluate the extent of thiol oxidative stress produced by 2-AAPA in cardiomyocytes, to compare the extent of thiol oxidative stress produced in this model to other pharmacologic oxidative stress models, to establish proof of concept that the GR inhibitor can be used to create thiol oxidative stress in medication screening using carvedilol as an example, and to explore the use of 2-AAPA in an in vivo model by determining the extent of thiol oxidative stress produced in mouse heart. The broad objective of this project is to create a model of thiol oxidative stress in cardiomyocytes. This model could be used in further research examining the pathophysiology of cardiovascular diseases and the impact that thiol alterations have on the development of these conditions. This model could also potentially be used in early screening of medications for cardiovascular conditions and to evaluate the effects of medications in thiol oxidative stress conditions at the molecular level. PUBLIC HEALTH RELEVANCE: Cardiovascular disease is a leading cause of morbidity and mortality in the United States. Several of the major cardiovascular disorders, including injury following a myocardial infarction, heart failure, and atherosclerosis, are associated with oxidative stress. This project seeks to establish a research model of thiol alterations in oxidative stress conditions that could be used to further study the development of cardiovascular disorders and to develop new treatment interventions.